We have focused on DNA complexes of restriction endonucleases in particular. Beyond their unparalleled importance as tools for the analysis and manipulation of DNA, restriction enzymes have proven remarkable model systems for studying numerous aspects of protein-nucleic acid interactions. These proteins combine high binding strength and extraordinary sequence specificity. [unreadable] Work is currently in progress on the type II restriction enzyme, EcoRV. Typically restriction endonucleases can distinguish between specific recognition and nonspecific DNA sequences quite efficiently. At present, however, results in literature suggest a very low sequence stringency for EcoRV. The majority of studies show only weak selectivity, below 10 for the relative specific-nonspecific competitive binding constant. Only one group has reported value of 120 at the same experimental conditions. X-ray crystal structures are available for both cognate and non-cognate EcoRV complexes. The interface of the specific complex is essentially anhydrous with many direct DNA-protein interactions and is very different from the non-cognate complex that has a large water filled gap at the protein-DNA interface. The substantial difference in structure would suggest a large difference in binding energy as is true for other type II restriction enzymes. [unreadable] We have applied our unique perspective and experimental tools to reexamine this nuclease. The novel self-cleavage assay we developed recently is broadly applicable to measuring DNA-protein interactions, particularly DNA binding of restriction endonucleases. This solution technique uses the cleavage reaction of restriction endonucleases to measure sensitively their binding to DNA. It has same sensitivity as gel mobility shift assay without its limitations. Additionally the self-cleavage assay does not require labeling DNA or protein with fluorophores that could perturb binding and that are typically sensitive to buffer conditions. We have shown that the self-cleavage assay can be used to quantitate EcoRV-DNA binding. After adding cleavage mix containing Mg2+, high enough concentrations of competing oligonucleotide that contains the recognition sequence, and neutral osmolytes, e.g., triethylene glycol or TMAO, to the pre-formed complex, only DNA fragments with initially bound enzyme are cleaved. [unreadable] Equilibrium measurements are meaningful only after it is known that the reaction time is sufficiently long to reach equilibrium. It is not unusual for proteins that have a high affinity for DNA to dissociate quite slowly from its specific sequences with half-life times measured in minutes and even hours. Association rate constants are usually much faster. Surprisingly, the association kinetics of EcoRV shows at least two components; one that is quite fast as is typical for specific binding. The other is unusually slow with a half-life time of about 20 min. This is far different from our previous observations with EcoRI. These results and the higher order complexes seen with the gel mobility shift assay suggest that EcoRV can form tetramers on DNA. We do not know if tetramers are preformed in solution or dimers meet on the DNA. We can distinguish the two by changing the relative and absolute concentrations of protein and DNA. The slow component of the association kinetics indicates that at least 2 hours incubation is necessary to reach equilibrium. The unusual on-rate kinetics for EcoRV could explain the puzzling literature results. It would be easy to underestimate amount of bound protein (and consequently binding constant) if the incubation time was not long enough to reach equilibrium. [unreadable] We have shown that the relative specific-nonspecific binding constant for EcoRV depends on water activity with a significant increase in binding specificity with increase of triethylene glycol concentration. We can determine the difference in sequestered water between the complexes through the dependence of the relative specific-nonspecific binding constant on solution osmotic pressure. Our preliminary estimation is that the nonspecific complex sequesters about 160 more water molecules than the specific complex. This value correlates quite well with the difference in volume of the interface cavities of specific and non-cognate complexes seen in the crystal structures. We have also estimated the relative specific-nonspecific binding constant as about 400, a value much larger than previously measured by others. As with other restriction nucleases, EcoRV specific sequence binding is stringent. [unreadable] We have also developed a method for stabilizing labile DNA-protein complexes for analysis by the gel mobility shift assay. Polyacrylamide gels stabilize DNA-protein or protein-protein complexes by a crowding or caging mechanism. Many nonspecific DNA-protein complexes, however, are weak enough that they do dissociate while electrophoresing giving smeared bands that are difficult to quantitate precisely. We find that adding osmolytes directly to the gel can further stabilize weak complexes. This project is a logical continuation of our previous work on trapping DNA-protein complexes. We now routinely use the stop-reaction protocol for all gel mobility shift assays. Since complex dissociation in the gel matrix is accompanied by a change in solvent accessible surface area (SASA), different osmolytes will have different efficacies depending on their extent of exclusion from the newly exposed surfaces. Experiments working with nonspecific complexes of EcoRI and BamHI show that triethylene glycol is particularly effective at inhibiting dissociation, does not interfere with normal gel polymerization, and does not significantly slow normal migration. Extension of this approach to other techniques for separating complex and free components as gel chromatography and capillary electrophoresis is straightforward.[unreadable] Work is now in progress to develop even better stabilizing conditions and fully eliminate in-gel dissociation. We intend to check variety of neutral solutes for their ability to stabilize weak DNA-protein in the gel.